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PubMed1991

Evidence for quantitative and qualitative differences in functional activation of Mls-reactive T cell clones and hybridomas by antigen or TcR/CD3 antibodies.

In this study, we demonstrated that some V beta 6+, CD4+, Mls-1a-specific T cell clones had cytolytic activity when stimulated with anti-T cell receptor(TcR)/CD3 monoclonal antibodies (mAb), but not with targets expressing Mls-1a, although they produced lymphokines (interleukin 2 and interferon-gamma) in response to both types of stimuli. To examine the possibility that lack of cytolysis resulted from expression of the Mls-1a antigen on merely a fraction of splenic B blasts, we (a) used the B cell lymphoma LBB.3.4.16 and (b) measured esterase secretion which is generally concurrent with cytotoxic T lymphocyte (CTL) activity. The B cell lymphoma maximally stimulated the T cell clone for interferon-gamma production when responding and stimulating cells were incubated at a 1:1 ratio, but it was never killed by the Mls-1a-specific T cell clone unless TcR/CD3-specific mAb were added. Furthermore, a fivefold excess of the Mls-1a B cell lymphoma did not induce any secretion of esterase, which was observed only in the presence of the TcR/CD3-specific mAb. Comparison of the reactivity of two Mls-1a-specific T cell hybridomas expressing the same TcR at similar surface density, revealed both quantitative and qualitative differences between CD3-specific mAb and Mls stimulation of the hybridomas. A small quantitative difference in the sensitivity of hybridoma FJ22.5 to stimulation with V beta 6 or CD3-specific mAb resulted in a marked decrease in efficiency of stimulation by Mls-1a for interleukin 2 production and to inability to detect growth inhibition by Mls-expressing cells. A qualitative difference was observed when analyses of inositol phosphate production were performed under optimal conditions of stimulation of the highly responsive T cell hybridoma (FJ8.1): only stimulation with CD3-specific mAb, but not Mls-expressing cells, could induce detectable inositol phosphate production. Lack of cytolysis of Mls-1a class II-expressing B cells may have evolutionary significance in view of the recent mapping of Mls to mouse mammary tumor virus genes.

Animals↗

PubMed1994

A human CD4 transgene rescues CD4-CD8+ cells in beta 2-microglobulin-deficient mice.

The specificity of the alpha beta T cell receptor for class I or class II major histocompatibility complex (MHC) molecules determines whether a mature T cell will be of the CD4-CD8+ or CD4+CD8- phenotype, respectively. We show here that a human CD4 transgene can rescue a significant fraction of CD4-CD8+ T cells in beta 2-microglobulin-deficient mice. Cells with this phenotype could be induced to become potent killers of targets expressing allogeneic MHC antigens, indicating that lineage commitment can precede the rescue of developing cells by the T cell receptor for antigen and the CD4 coreceptor.

Animals↗

PubMed1990

Hyperplasia of mouse duodenal crypts and its control by NK cells during the initial phase of DMH carcinogenesis.

The possible regulatory role of NK cells on early events in chemical carcinogenesis remains undefined. The present study examined whether NK cells control 1,2-dimethylhydrazine (DMH)-induced hyperplasia of the duodenal crypt in CD1 mice. Mice receiving chronic DMH treatment showed a dose-dependent hyperplasia confined to the proliferative zone, with a parallel increase in mitotic and 3H-TdR-labelled cells and significant suppression of splenic NK activity. Complete ablation of splenic NK activity with anti-asialo GM-I antibody (alpha AGM-I) treatment slightly enhanced hyperplasia. Halving of the DMH dose for 2 weeks led to regression of hyperplasia, which was totally prevented by alpha AGM-I treatment. The alpha AGM-I treatment alone did not influence crypt size in normal mice. Finally, a stimulation of NK activity with Poly I:C treatment in DMH-treated mice caused regression of the DMH-induced hyperplasia. Our results suggest that hyperplastic cells with possible genetic alterations induced by the carcinogen express target structures for NK cells, but that simultaneous carcinogen-induced suppression of NK activity hampers their containment, allowing progression of hyperplasia to neoplasia, possibly owing to additional genetic changes.

1,2-Dimethylhydrazine↗

PubMed2003

Aspartylglucosaminidase (AGA) is efficiently produced and endocytosed by glial cells: implication for the therapy of a lysosomal storage disorder.

BACKGROUND: Aspartylglucosaminuria (AGU) represents diseases affecting the central nervous system and is caused by a deficiency of a lysosomal enzyme, aspartylglucosaminidase (AGA). AGA, like lysosomal enzymes in general, are good targets for gene therapy since they move from cell to cell using the mannose-6-phosphate receptor. Consequently, only a minority of target cells need to be corrected. Here, we wanted to determine which cell type, neurons or glia would better produce AGA to be transported to adjacent cells for use in possible treatment strategies. METHODS: Adenoviruses containing tissue-specific glial fibrillary acidic protein (GFAP) promoter and neuron-specific enolase (NSE) promoter were generated to target expression of AGA in Aga-deficient mouse primary glial and neuronal cell cultures. In addition an endogenous AGA promoter was used. The experimental design was planned to measure the enzymatic activities in the cells and media of neurons and glia infected with each specific virus. The endocytosis of AGA was analyzed by incubating neuronal and glial cells with media produced by each virus-cell combination. RESULTS: AGA promoter was shown to be a very powerful glia promoter producing 32 times higher specific AGA activity in glia than in neurons. GFAP and NSE promoters also produced a clear overexpression of AGA in glia and neurons, respectively. Interestingly, both the NSE and GFAP promoters were not cell-specific in our system. The amount of exocytosed AGA was significantly higher in glial cells than neurons and glial cells were also found to have a greater capacity to endocytose AGA. CONCLUSIONS: These data indicate the importance of glial cells in the expression and transport of AGA. Subsequently, new approaches can be developed for therapeutic intervention.

Adenoviridae↗

PubMed1993

Induction of epidermal hyperplasia, hyperkeratosis, and papillomas in transgenic mice by a targeted v-Ha-ras oncogene.

The regulatory elements of the human keratin K1 gene have been used to target expression of the v-Ha-ras oncogene exclusively in the epidermis of transgenic mice. We developed 12 transgenic mouse lines that express the HK1.ras transgene, producing epidermal hyperplasia in neonates and hyperkeratosis in juveniles. Eventually this skin phenotype diminished but with time adult animals developed papillomas that could persist or regress. The rate and frequency of tumorigenesis appeared to be limited, which suggests that v-Ha-ras requires a second or even third event to elicit and maintain a benign phenotype in transgenic mice. Since in certain transgenic lines papillomas appeared at wound sites, it appears that the promotion stimulus from wounding may be a second event. We envision that such transgenic mice that express v-Ha-ras in the epidermis will become a powerful model for assessing how environmental and molecular factors affect the process of multistage skin carcinogenesis in vivo, as well as a model for evaluating novel therapeutic protocols.

Aging↗

PubMed2002

Inactivation of olfactory sensilla of a single morphological type differentially affects the response of Drosophila to odors.

The olfactory organs on the head of Drosophila, antennae and maxillary palps, contain several hundred olfactory hairs, each with one or more olfactory receptor neurons. Olfactory hairs belong to one of three main morphological types, trichoid, basiconic, and coeloconic sensilla, and show characteristic spatial distribution patterns on the surface of the antenna and maxillary palps. Here we show that targeting expression of the cell-death gene reaper to basiconic sensilla (BS) causes the specific inactivation of most olfactory sensilla of this type with no detectable effect on other types of olfactory sensilla or the structure of the antennal lobe. Our data suggest that BS are required for a normal sensitivity to many odorants with a variety of chemical structures, through a wide range of concentrations. Interestingly, however, in contrast to other odorants tested, the behavioral response of ablated flies to intermediate concentrations of propionic and butyric acids is normal, suggesting the involvement of sensilla unaffected by ectopic reaper expression, probably coeloconic sensilla that respond strongly to these two organic acids. As inactivation of BS causes an underestimation of the concentration of both acids detectable at both the highest and lowest odorants concentrations, our results suggest that concentration coding for these two odorants relies on the integration of signals from different subsets of sensilla, most likely of different morphological types.

Animals↗

PubMed2006

Distinct functions of neuronal synaptobrevin in developing and mature fly photoreceptors.

Neuronal synaptobrevin (n-Syb, alias VAMP2), a synaptic vesicle membrane protein with a central role in neurotransmission, is specifically cleaved by the light chain of tetanus neurotoxin (TNT) that is known to reliably block neuroexocytosis. Here, we study fly photoreceptors transmitting continuous, graded signals to first order interneurons in the lamina, and report consequences of targeted expression of TNT in these cells using the UAS/GAL4 driver/effector system. Expressing the toxin throughout photoreceptor development causes developmental, electrophysiological, and behavioral defects. These can be differentiated by confining toxin expression to shorter developmental periods. Applying a method for controlled temporal and spatial TNT expression, we found that in the early pupa it impaired the development of the retina; in the midpupa, during synapse formation TNT caused a severe hypoplasia of the lamina that persisted into adulthood and left the photoreceptor-interneuron synapses of the lamina without function. Finally, during adulthood TNT neither blocks synaptic transmission in photoreceptors nor depletes the cells of n-Syb. Our study suggests a novel, cell type-specific function of n-Syb in synaptogenesis and it distinguishes between two synapse types: TNT resistant and TNT sensitive ones. These results need to be taken into account if TNT is used for neural circuit analysis.

Animals↗

PubMed1998

Genes for sexual behavior.

The mating behavior of Drosophila melanogaster is a stereotyped sequence of fixed action patterns, composed of orientation, tapping, singing, licking, attempted copulation and copulation. Mutations that block a unique aspect of mating behavior were isolated and analyzed at the cellular and molecular levels. The wild-type counterparts of the mutated genes were shown to rescue the phenotypes by their ubiquitous or targeted expression in some of the mutants. This strategy of artificial control of fly behavior opens up an avenue for studies to identify the neural center for individual behavioral actions.

Animals↗

PubMed1999

Maspin plays an important role in mammary gland development.

Maspin is a unique member of the serpin family, which functions as a class II tumor suppressor gene. Despite its known activity against tumor invasion and motility, little is known about maspin's functions in normal mammary gland development. In this paper, we show that maspin does not act as a tPA inhibitor in the mammary gland. However, targeted expression of maspin by the whey acidic protein gene promoter inhibits the development of lobular-alveolar structures during pregnancy and disrupts mammary gland differentiation. Apoptosis was increased in alveolar cells from transgenic mammary glands at midpregnancy. However, the rate of proliferation was increased in early lactating glands to compensate for the retarded development during pregnancy. These findings demonstrate that maspin plays an important role in mammary development and that its effect is stage dependent.

Animals↗

PubMed2000

Recombinant Nup153 incorporates in vivo into Xenopus oocyte nuclear pore complexes.

Nup153 is a molecular constituent of the nuclear basket of the nuclear pore complex (NPC) that plays a critical role in nuclear export of RNAs and proteins. In an effort to map this nucleoporin more precisely within the nuclear basket we have developed an experimental approach for localizing Nup153 expressed and incorporated in vivo into Xenopus oocyte NPCs. This approach involves the microinjection into the cytoplasm of Xenopus oocytes of in vitro synthesized mRNA from a vector encoding an epitope-tagged cDNA. Here we present results obtained by Western blots, fluorescence microscopy, and immuno-electron microscopy, which clearly document that the heterologous protein is properly expressed, targeted, and incorporated into preexisting Xenopus NPCs. This new approach for localizing nucleoporins within the structure of the NPC overcomes limitations of previous techniques and allows for greater specificity and resolution than have been possible with previous methods.

Animals↗

PubMed1996

Recognition of human cytomegalovirus gene products by HCMV-specific cytotoxic T cells.

The antigen specificity of HCMV-specific cytotoxic T cells (CTL) was investigated in healthy adult seropositive donors by analyzing the frequency of CTL precursors (CTLp) against six different HCMV-encoded proteins. HCMV-specific, CD8+ CTL clones from two donors were also characterized. Thje precursor frequency analysis showed that a major phosphoprotein of HCMV, pp65 (UL83), was the immunodominant CTL target antigen in healthy seropositive adults. The frequency of CTLp specific for different HCMV proteins ranged from 1/12,000 for pp65 to > 1/100,000 for gB (UL55), gH (UL75), pp50 (UL44), and pp28 (UL98). Characterization of the HCMV-specific CTL clones from two donors revealed that the majority of clones lysed targets expressing pp65 and that none of the clones recognized other HCMV proteins.

Antigens, Viral↗

PubMed2000

Design and testing of ribozymes for cancer gene therapy.

This chapter describes procedural aspects for development of ribozymes in general, and specifically, that cleave mRNA to an essential cellular gene, the AC40 subunit of RNA pol I. Ribozyme design includes functional selection of binding sites followed by computer modeling. These ribozymes are being used in vectors that target expression to the prostate via tissue specific promoters (Voeks, Norris, and Clawson, 1998) and have demonstrated efficacy.

Animals↗

PubMed1992

Two subtypes of HLA-B51 differing by substitution at position 171 of the alpha 2 helix.

Newly defined antigens of the B5, B35 cross-reacting group have been found in Japanese and North American Indians. Nucleotide sequencing of the alleles encoding the Japanese B5.35 antigen and the variant B5 antigen from the Piman Indians show them to be identical. This new allele, B*5102, differs from B*5101 by a single nucleotide substitution that changes residue 171 from histidine to tyrosine. Residue 171, which is part of the alpha 2 helix, is believed to contribute directly to peptide interaction in the A pocket of the binding groove and is either histidine or tyrosine in all HLA-A, B, C heavy chains. Tyrosine 171 is shared by B*5102, B*3501, B*3502, and B*5301 and must be responsible for the serological cross-reactivities of these molecules not shared with B*5101. Stimulation of lymphocytes from a B*5101 positive donor with B*5102 positive cells failed to generate cytotoxic T cells with specificity for the difference between these molecules. However, one out of five clones of cytotoxic T cells raised against B*5101 failed to lyse targets expressing B*5102. Substitution of histidine for tyrosine at residue 171 affected recognition of HLA-B35-restricted human minor histocompatibility antigen-specific T cell clones.

Amino Acid Sequence↗

PubMed1988

SV40 T-antigen is a histocompatibility antigen of SV40-transgenic mice.

Although the extensive family of non-H-2 histocompatibility (H) antigens provides a formidable barrier to transplantation, the origin of their encoding genes are unknown. Recent studies have demonstrated both the linkage between H genes and retroviral sequences and the ability of integrated Moloney-murine leukemia virus to encode what is operationally defined as a non-H-2 H antigen. The experiments described in this communication reveal that skin grafts from an SV40 T-antigen transgenic C57BL/6 mouse strain are rejected by coisogenic C57BL/6 recipients with a median survival time of 49 days, which is comparable to those of many previously defined non-H-2 H antigens. The specificity of this response for SV40 T-antigen was demonstrated by the identification of SV40 T-antigen-specific cytolytic T lymphocytes and antibodies in multiply-grafted recipients. Although these cytolytic T lymphocytes could detect SV40 T-antigen on syngeneic SV40-transformed fibroblasts, they neither could be stimulated by splenic lymphocytes from T-antigen transgenics nor could they lyse lymphoblast targets from T-antigen transgenics. These observations suggest a limited tissue distribution of SV40 T-antigen in these transgenics. These results confirm the role of viral genes in the determination of non-H-2 histocompatibility antigens by the strict criteria that such antigens stimulate (1) tissue graft rejection and (2) generation of cytolytic T lymphocytes. Furthermore, they suggest that the SV40 enhancer and promoter region can target expression of SV-40 T-antigen to skin cells of transgenic animals.

Animals↗

PubMed1994

From in vitro to in vivo. Progress in the use of cultured cells for human therapy.

The use of cultured cells with the ultimate goal of using the cells or their products for human therapy has experienced an exponential growth during the last decade. Stable cell cultures have been established and genetically modified to obtain high quality products for protein replacement therapy or vaccines. Cells have also been directly isolated from the human organism and, after their expansion in vitro, been retransferred as skin grafts for treatment of burns or for cancer therapy by activated lymphocytes. With the explosive development of molecular biology techniques, it is now possible to genetically modify ex vivo, cells derived from the human body. These modifications should allow targeted expression of therapeutic genes into specific cells which will, upon retransfer to the body, exert their therapeutic action in a diseased organism.

Animals↗

PubMed1995

Production of human surfactant protein C in milk of transgenic mice.

Respiratory distress syndrome (RDS), caused by lack of pulmonary surfactant, affects 65 000 infants annually in the USA. Surfactant replacement therapy reduces the morbidity and mortality associated with RDS. Human surfactant protein C (SP-C) is an important component of pulmonary surfactant. To produce human SP-C, a construct using the rat whey acidic protein (WAP) promoter and 3' untranslated regions to target expression of the human SP-C gene to the mammary gland of transgenic mice was created. WAP/SP-C mRNA expression was detected in all transgenic lines analysed. SP-C was expressed in a copy-number-dependent and integration-site-independent fashion, with levels of expression ranging from 0.01% to 36.0% of the endogenous mouse WAP mRNA, and WAP/SP-C mRNA expression levels were greater than those of of the endogenous mouse lung SP-C mRNA. Expression at the RNA level was specific to the mammary gland and paralleled the endogenous WAP expression pattern during mammary gland development. Expression and secretion of the SP-C protein in the lactating mammary gland was demonstrated by western blots performed on whole milk using an anti-SP-C polyclonal antibody. Immunoreactive proteins of MW 22 and 12-14 kDa appeared only in transgenic milk. The 22 kDa protein represents the proprotein, and the 12-14 kDa is a processed form of SP-C.

Animals↗

PubMed1985

Tumor growth and evasion of immune destruction: UV-induced tumors as a model.

A number of complex interrelating mechanisms contributing to progressive growth of an immunogenic tumor and its evasion of immune destruction are exemplified in the model of the UV-induced tumor 1591 (fig. 3). The effects of (i) carcinogen-induced immune suppression, (ii) age-related changes in immune competence, (iii) generation and immunoselection of tumor cell variants which do not express target tumor antigen and (iv) tumor-induced immune suppression have been documented. The findings may help to assess the problems and the possibilities of immunotherapy of cancer. The problems reside in the many avenues of escape available to the tumor. The possibilities for immunotherapy derive from the fact that progressively growing tumors may still retain immunogenic antigens and therefore be susceptible to immune attack.

Aging↗

PubMed1999

Protective antitumor activity through dendritic cell immunization is mediated by NK cell as well as CTL activation.

Dendritic cells (DCs) are potent professional antigen-presenting cells (APC) capable of inducing the primary T cell response to antigen. Although tumor cells express target antigens, they are incapable of stimulating a tumor-specific immune response due to a defect in the costimulatory signal that is required for optimal activation of T cells. In this work, we describe a new approach using tumor-DC coculture to improve the antigen presenting capacity of tumor cells, which does not require a source of tumor-associated antigen. Immunization of a weakly immunogenic and progressive tumor cocultured with bone marrow-derived DCs generated an effective tumor vaccine. Immunization with the cocultured DCs was able to induce complete protective immunity against tumor challenges and was effective for the induction of tumor-specific CTL (cytotoxic T lymphocyte) activity. Furthermore, high NK cell activity was observed in mice in which tumors were rejected. In addition, immunization with tumor-pulsed DCs induced delayed tumor growth, but not tumor eradication in tumor-bearing mice. Our results demonstrate that coculture of DCs with tumors generated antitumor immunity due to the NK cell activation as well as tumor-specific T cell. This approach would be useful for designing tumor vaccines using DCs when the information about tumor antigens is limited.

Animals↗